Internalisation of virus into cells

ABSTRACT

A chimeric transmembrane protein which promotes viral entry into cells comprises: (i) an extracellular domain capable of binding a virus; and (ii) an intracellular internalisation signal.

FIELD OF THE INVENTION

[0001] The present invention relates to viral infection. In particularthe invention relates to a protein designed to promote viral entry intocells, cells and a non-human transgenic animal comprising such a proteinand a method of infecting such cells and animals with a virus. Theinvention also relates to methods for testing and screening anti-viralagents.

BACKGROUND OF THE INVENTION

[0002] In order for a virus to establish an infection, it must firstenter a cell. Human immunodeficiency virus type 1 (−1) infection, forexample, involves binding of the viral envelope protein gp120/160 tocell surface CD4 molecules followed by interactions with a coreceptor.This results in fusion of the viral and cellular membranes. HIV-1 virusuptake can be reconstituted in heterologous cell lines by theco-expression of CD4 and the respective chemokine receptor, suggestingthat cell-type and species specific infection is largely determined atthe level mediated by the viral receptors.

[0003] The mechanisms by which other viruses enter cells are less wellunderstood and no proper cell-based systems are available. For example,hepatitis C virus (HCV) is thought to bind to CD81 receptors expressedon the cell surface of hepatocytes via the structural protein E2,although the role of CD81 in mediating viral entry is controversial asCD81 is widely expressed on cell surfaces and thus, cannot explain virustropism to hepatocytes. Moreover; in cell fusion assays that usechimeric HCV envelope proteins, over-expression of human CD81 has beenshown not to affect cell fusion activity. For many viruses, the cellularreceptors are even less well characterized.

[0004] The process of receptor-mediated endocytosis, by which cellsinternalize their plasma membrane together with molecules bound to cellsurface receptors, have been implicated as the route of cell entry byseveral viruses including rabies, herpes, Semliki Forest, African swinefever and HIV viruses. However, it has been reported that CD81 has apoor internalization efficiency and this may be one of the reasons whyCD81-overexpressing cells are only moderately permissive and can not bereproducibly infected by HCV.

[0005] Constitutively cycling receptors such as the transferrin receptor(TFR) and low density lipoprotein receptor (LDLR) are constitutivelyclustered in coated pits and can be rapidly internalized, transported tothe acidic endosome and finally recycled back to the cell surface. Theconstitutive internalisation of such receptors is mediated byinternalisation signals in their cytoplasmic domains. Theseinternalisation signals are self-determined structural motifs that mayconfer recycling properties to proteins that are not normallyendocytosed.

SUMMARY OF THE INVENTION

[0006] The present invention is based on the finding that a chimericprotein comprising an extracellular domain capable of binding to a virusand an intracellular internalisation domain enables virus to enter cellsin which it is expressed and thus enables viral infection to beestablished. In particular, the present inventors have engineeredendocytosis and membrane anchoring signals into the C-terminus of theimmunoglobulin heavy chain and have shown that these chimeric antibodiesare displayed on the cell surface and undergo endocytosis. The inventorshave shown that these cell surface antibodies can bind HIV-1 virus withhigh affinity which binding results in the internalization of the virusinto a human kidney cell line.

[0007] In addition, the present inventors have constructed two CD81chimeric receptors by linking either the N or C-terminus of CD81 withcytoplasmic domains of the transferrin receptor (TFR) or the low densitylipoprotein receptor (LDLR), respectively and have found that the CD81chimeras have better internalization efficiency than wild-type CD81.Further, the inventors have shown that the internalization efficienciesof these receptors is correlated with infectivity of cultured livercells that are over-expressing either wild-type or chimeric CD81receptors by HCV virions produced by a tetracycline-inducible cellculture system.

[0008] Cells and non-human transgenic animals expressing such chimericproteins are useful in methods for identifying novel pharmaceuticalagents. These agents may be used in the therapeutic and/or prophylactictreatment of viral infections.

[0009] Accordingly, the present invention provides:

[0010] a chimeric transmembrane protein comprising:

[0011] (i) an extracellular domain capable of binding a virus; and

[0012] (ii) an intracellular internalisation signal;

[0013] a polynucleotide encoding a protein of the invention;

[0014] a vector comprising a polynucleotide of the invention;

[0015] a cell comprising a protein, polynucleotide or vector of theinvention;

[0016] a cell comprising a protein of the invention, which cell isinfected with a virus that is capable of binding to said protein;

[0017] a transgenic non-human animal comprising a cell of the invention;

[0018] a method for identifying an anti-viral agent, said methodcomprising:

[0019] (i) providing a cell expressing a protein of the invention or anon-human transgenic animal comprising a cell expressing a protein ofthe invention, which cell or animal is infected with a virus;

[0020] (ii) contacting said cell or animal with a test agent; and

[0021] (iii) monitoring viral infection;

[0022] thereby determining whether the test agent has anti-viralactivity;

[0023] a method for identifying an anti-viral vaccine or agent capableof preventing or inhibiting viral infection, which method comprises:

[0024] (i) providing a cell expressing a protein of the invention or anon-human transgenic animal comprising a cell expressing a protein ofthe invention;

[0025] (ii) contacting said cell or animal with a test agent;

[0026] (iii) contacting said cell or animal with a virus capable ofbinding to said protein; and

[0027] (iv) determining whether the test agent prevents or limits viralinfection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1A is a schematic diagram showing the membrane topologies ofwild-type CD81, transferrin receptors (TFR) and low density lipoproteinreceptors (LDLR), and the predicted topologies of CD81 chimeras. σ and νrepresent cytoplasrnic domains of LDLR and TFR, respectively, that werefused to CD81 to form chimeric receptors.

[0029]FIG. 1B shows the results of FACScan analysis of the surfaceexpression of CD81, transferrin and LDL receptors in parental Huh7 andstably transfected Huh7 clones. Cells were stained with specificantibodies (anti-CD81, anti-TFR or anti-LDLR), followed byphycoerythrin-conjugated goat anti-mouse IgG (unfilled histograms).Filled histograms represented staining by phycoerythrin-conjugatedantibody only. 293T cells expressing endogenous receptors were used aspositive controls.

[0030]FIG. 2 shows the results of western analysis of theinternalization of anti-CD81 antibodies via wild-type and chimeric CD81.Total IgG in the cell lysate represents both surface-bound andinternalized antibody after living cells were allowed to endocytoseantibody for 1 h at 37° C. Only internalized IgG was left in the celllysate after an acid wash was used to remove all surface-bound antibody.Expression of intracellular heat shock 70 protein, Hsp70, was used fornormalization.

[0031]FIG. 3 shows the rate of down modulation and re-expression of bothwild-type and chimeric CD81 receptors on the cell surface of Huh7 stableclones as determined by FACScan analysis. Surface expression of CD81receptors was determined after the cells were incubated with ananti-CD81 antibody at 37° C. for different times, washed extensively andincubated with anti-CD81 antibody and phycoerythrin-conjugated goatanti-mouse IgG. Mean fluorescence was then computed for each time-point.

[0032]FIG. 4A shows that under non-reducing conditions, E2 exists mainlyas high molecular weight aggregates with only a small percentageexisting as monomers. In the presence of reducing agent, all aggregatesdissociated to form monomers. Truncated E2 protein was expressed in 293Tcells and detected using an anti-myc antibody as the protein was fusedto a myc-tag at the C-terminal. Control cells were transfected withvector only.

[0033]FIG. 4B illustrates the binding of truncated E2 protein to Huh7stable clones overexpressing wild-type or chimeric CD81 which wasobserved after E2 protein was overlaid onto monolayers of cells at 37°C. for 4 h. No binding of E2 to untransfected Huh7 cells was observed.Beta-actin expression showed equal loading of total cell lysate.

[0034]FIG. 5 shows the results of RT-PCR which was performed to assayfor the presence of viral transcripts in the culture media from Huh7cells stably expressing an inducible full length HCV genome. PositiveRT-PCR products were observed in culture media from cells that had beeninduced with tetracycline for 5 days (after antibody capture withanti-E2 antibody) and these products were still observed after treatmentwith DNaseI and RNaseA. No product was observed if the cells were notinduced with tetracycline.

[0035]FIG. 6 shows the results of the RT-PCR which was performed toassay for the presence of viral transcripts in Huh7 stables clones afterthey were exposed to infectious media. Huh7-CD81WT showed a low amountof viral transcript while none was detected in parental Huh7 cells.Huh7-CD81-ΔLDLR and Huh7-CD81-ΔTFR expressing cells exhibited increasedlevels of both plus and minus strand viral RNA compared to Huh7-CD81WT,indicating an increase in viral uptake and replication in these cells.Cellular GAPDH was used for normalization and positive control was doneusing RNA from cells transfected with the HCV full-length genome.

[0036]FIG. 7A shows the results of viral infection experiments. Lanes 2to 4: No viral transcripts were observed in Huh7-CD81WT cells overlaidwith infectious media that has been pre-cleared with an anti-E2antibody, while viral transcripts were observed when the cells wereoverlaid with infectious media not pre-cleared or pre-cleared withcontrol anti-c-myc antibody. Lanes 5 and 6: No viral transcripts wereobserved in Huh7-CD81WT cells when the cells were pre-incubated with ananti-human CD81 antibody (anti-hCD81) before the addition of infectiousmedia On the other hand, pre-incubation with an anti-mouse CD81 antibody(anti-mCD81) did not affect the infection of Huh7-CD81WT cells. Anegative control was performed using cells that were overlaid withnon-infectious media.

[0037]FIG. 7B shows the results of blocking experiments performed onHuh7-CD81-ΔTFR cells. Pre-clearing with anti-E2 prevented infection(lane 2 to 4). However, infection of Huh7-CD81-ΔTFR was not blocked byanti-human CD81 antibody (lane 5) presumably because of the higherinternalization efficiency of CD81-ΔTFR chimeric receptor compared towild-type CD81.

[0038]FIG. 8 shows the partial sequences of IgH (A) and IgL (B) chainsisolated from hybridoma 902 showing the open reading frame and thevariable regions. N-terminal sequences of the mature proteins determinedby Edman sequencing are underlined.

[0039]FIG. 9A shows the results of western analysis indicating that IgHand IgL associated with each other. Culture medium (containingsecreted/extracellular proteins) and cell lysate (intracellular proteinreleased by cell lysis) from transiently transfected cells wereincubated with protein A/G beads and bound IgH and IgL were detected bywestern analysis. Cells transfected with IgL and IgH secreted bothchains into the culture medium and they were associated with each othersince capturing IgH with protein A/G beads also pulled down IgLproteins. For cells transfected with IgL and chimeric IgH (IgH-ΔCI-M6PRor IgH-ΔLDLR), the heavy chains were found intracellularly, indicatingthat they were associated with the membranes.

[0040]FIG. 9B shows the results of FACScan analysis illustrating thatcells transiently transfected with IgL and IgH did not express antibodyon the cell surface, as the mean fluorescence of the cells was the sameas untransfected cells. In contrast, cells transfected with IgL andchimeric IgH (IgH-ΔCI-M6PR or IgH-ΔLDLR) showed high mean fluorescence,indicating that the antibodies were expressed on the cell surface.

[0041]FIG. 9C shows the expression of heavy and light chain antibodiesin cell lysates of stable clones of 293 cells transfected with IgL andIgH-ΔCI-M6PR or IgH-ΔLDLR.

[0042]FIG. 10 illustrates the results of western analysis to detectgp120 present in HIV-1 MC99IIIBΔTat-Rev virus that had beenimmunoprecipitated using antibodies secreted into culture medium byhybridoma 902 or by 293T cells transiently transfected with IgL and IgHcaptured onto protein A/G beads. No virus was detected when culturemedium from cells transfected with IgH only was used.

[0043]FIG. 11A shows the expression of chimeric antibodies on the cellsurfaces of stable clones of 293 cells transfected with IgL andIgH-ΔCI-M6PR (clone 9) or IgH-ΔLDLR (clone 10) as determined by FACScananalysis using an anti-mouse FITC-conjugated F(ab′)₂ antibody (unfilledhistograms). Filled histograms represented unstained cells.Untransfected cells were used as control.

[0044]FIG. 11B shows PMA-induction of luciferase activities in stablytransfected 293-IgH-ΔCI-MPR (clone 9) cells after infection with thepseudotype-virus, HIV strain HBX2 (He and Landau, J.Virol. 69,4587-4592, 1995), which contains a luciferase reporter gene insertedinto the nef gene. Infected 293-IgH-ΔCI-MPR cells that were not treatedwith PMA did not show luciferase activity. Neither untransfected (UNT)nor 293-IgH-ΔLDLR (clone 10) cells showed significant luciferaseactivity.

[0045]FIG. 12 shows the internalization and replication of HCV inHuh7-TfR-CD81 cells. Nested RT-PCR was performed to assay for thepresence of viral transcripts in Huh7 stables clones after they wereexposed to sera from two HCV-infected patients (A and B). Cellular GAPDHmRNA was used to check that similar number of cells was used in theexperiments (panel H). (A) After the cells were exposed to serum frompatient A, a significant amount of positive strand HCV RNA was detectedin Huh7-TfR-CD81 cells but not in untransfected Huh7 and Huh7-CD81WTcells (panel I, positive strand RT-PCR products visualized under UVlight after ethidium bromide staining). Negative strand HCV RNA was alsodetected only in Huh7-TfR-CD81 cells (negative strand RT-PCR productsvisualized under UV light after ethidium bromide staining panel (III)and analyzed by Southern blot with a specific probe (panel IV)). (B) Nopositive or negative strand viral transcripts were detected inuntransfected Huh7, Huh7-CD81WT or Huh7-TfR-CD81 cells when serum frompatient B was used directly to overlay onto the cells (withouttreatment, lanes 1-3, panel I, III and IV). When the serum waspre-treated with 0.05% deoxychloate before overlaying on the cells,positive strand RT-PCR products was observed in Huh7-TfR-CD81 but not inuntransfected Huh7 and Huh7-CD81WT cells (0.05% deoxychloate, lanes 4-6,panel I). Negative strand RT-PCR can be detected under UV light afterethidium bromide staining (panel III) and by Southern blot analysis(panel IV), only for Huh7-TfR-CD81 cells. (C) Positive strand RT-PCRproducts from Huh7-TfR-CD81 cells that were overlaid with the serum frompatient A or B, were ligated into pCRII-TOPO vector and sequenced fromboth directions. Sequences were aligned and compared with thecorresponding 5′ non-coding region from representative isolates, HCV-BKfor genotype 1b (Takamizawa et al, J. Virol 65, 1105-1113, 1991; GenBankaccession number M58335) and HCV-1 for genotype 1a (Choo et al., PNASUSA 88, 2451-2455, 1991; GenBank accession number M62321). Nucleotidesmatching to HCV-BK are represented by and mismatches are boxed.Nucleotide numberings shown on the left refer to the nucleotidepositions in HCV-BK.

[0046]FIG. 13 shows that internalization of HCV particles was abolishedwhen patient serum was pre-cleared with an anti-HCV E2 antibody. Afterthe overlay experiments shown in FIG. 12A, the remaining serum frompatient A was refrozen at −80° C. and later, thawed again for theseexperiments. (A) Serum was immunoprecipated with an anti-HCV E2 antibodyor anti-c-myc antibody (negative control) and protein A/G beads. Afterwashing, RNA was extracted from the immuno-complexes and analyzed atdifferent dilutions for the presence of positive strand HCV RNA. SomeHCV RNA were immunoprecipated by both antibodies (neat, lanes 2 and 6)but significantly (10-30×) more HCV RNA were bound to anti-E2 antibody(lanes 6 to 9) than anti-c-myc antibody (lanes 2 to 5). RT-PCR productswere still detected when the RNA immunoprecipated by anti-E2 antibodywas diluted 10× and 30× but no RT-PCR products observed for anti-c-mycantibody at these dilutions. DNA marker was loaded in lane 1 (bprepresents base pairs). (B) RT-PCR was performed to assay for thepresence of positive strand viral transcripts in Huh7 stables clonesafter they were exposed to serum from patients A. Positive strand RT-PCRproducts were observed in Huh7-TfR-CD81 (lane 4) and Huh7-CD81-LDLR(lane 1) cells but not in untransfected Huh7 (lane 2) or Huh7-CD81WTcells (lane 3). No positive strand RT-PCR products was observed inHuh7-TfR-CD81 cells overlaid with serum that was pre-cleared with ananti-HCV E2 antibody (lane 5), while positive strand RT-PCR productswere observed when the cells were overlaid with serum that waspre-cleared with control anti-c-myc antibody (lane 6).

DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention provides a chimeric transmembrane proteincomprising or, in some embodiments, consisting essentially of:

[0048] (i) an extracellular domain capable of binding a virus; and

[0049] (ii) an intracellular internalisation signal.

[0050] The protein may comprise a single polypeptide or may comprise twoor more associated polypeptides. Preferably, the protein comprises asingle polypeptide or two associated polypeptides.

[0051] Where the protein comprises two or more polypeptides, one or moreof the polypeptides may comprise a transmembrane domain. Theextracellular domain and the intracellular internalisation signal may bepart of the same polypeptide or may be present on associatedpolypeptides. Association of two or more polypeptides typically resultsin the constitutive internalisation of all associated polypeptides.

[0052] Extracellular Domain

[0053] An extracellular domain is capable of binding a virus. The virusmay be a DNA virus or a RNA virus. It may be a single-stranded virus ora double-stranded virus. The virus may be a flovivirus, picomavirus,myxovirus or herpes virus.

[0054] Typically binding of the extracellular domain to a virus occursby interaction of the extracellular domain with a viral protein.Generally, this viral protein will be expressed on the cell-surface ofthe virus. For example, where the virus is HIV the extracellular domainof the protein is typically capable of binding to gp120/160. Where thevirus is HCV, the extracellular domain is typically capable to bindingto E2. Where the virus is influenza A virus, the extracellular domain istypically capable of binding to hemagglutinin. Where the virus is herpessimplex virus, the extracellular domain is typically capable of bindingto glycoprotein C. Where the virus is a rhinovirus, the extracellulardomain is typically capable of binding to external proteins VPs. Wherethe virus is Epstein-Barr virus, the extracellular domain is typicallycapable of binding to glycoprotein gp350.

[0055] The ability of the extracellular domain to bind to a virus can bedetermined using any suitable assay. Typically, the ability of theextracellular domain to bind to a virus will be determined by monitoringbinding of the extracellular domain to a viral protein. Numerous methodsfor monitoring protein/protein interactions are known in the art, suchas a co-precipitation, co-purification and overlay assays.

[0056] The extracellular domain may comprise an antibody, or a fragmentthereof. Preferably, the extracellular domain comprises a fragment of anantibody, which fragment is capable of specifically binding to a viralprotein. An antibody, or other protein, “specifically binds” to aprotein when it binds with preferential or high affinity to the proteinfor which it is specific but does not substantially bind, not bind, orbinds with only low affinity to other proteins. A variety of protocolsfor competitive binding or immunoradiometric assays to determine thespecific binding capability of an antibody are well know in the art (seefor example Maddox et al, J. Exp. Med. 158,1211-1226, 1993). Suchimmonoassays typically involve the formation of complexes between thespecific protein and its antibody and the measurement of complexformation. Suitable antibody fragments include Fv, Fab and Fab′fragments and single-chain antibodies.

[0057] Preferably the antibody fragment capable of binding to a viruscomprises a variable region of a heavy chain and a variable region of alight chain. Typically the heavy and light chains are associated suchthat they form a binding site for a viral protein.

[0058] Preferably the heavy chain fragment is fused to a transmembranedomain. Typically, the light chain fragment has no transmembrane domainand is associated with the heavy chain via non-covalent protein-proteininteractions. Generally, the heavy chain and light chain will beassociated by means of a disulphide bond. It is therefore preferred thatthe fragments of the light and heavy chains both contain one or morecysteine residues that form disulphide bonds in the whole antibodymolecule.

[0059] The extracellular domain may comprise a cell surface receptor ora fragment thereof capable of binding to a virus. Any cell surfaceprotein which recognises and binds to a virus, preferably to a specificprotein on the surface of the virus may form the extracellular domain.For example where the virus is HIV the cell surface receptor is CD4 andwhere the virus is HCV the cell surface receptor is CD81, where thevirus is influenza A virus the receptor is sialic acid, where the virusin Herpes simplex virus the receptor is glycosaminoglycan heparansulphate, where the virus is Rhinovirus the receptor is ICAM-1 and wherethe virus is Epstein-Barr virus the receptor is C3d. A protein of theinvention may comprise-intracellular and transmembrane regions from acell surface receptor in addition to extracellular regions from thereceptor.

[0060] Intracellular Domain

[0061] A transmembrane protein of the invention comprises anextracellular internalisation signal. The term internalisation signalrefers to a region of a polypeptide which interacts with the endocytoticmachinery in a cell such that a protein comprising this polypeptideregion is constitutively internalised by endocytosis when present on thecell surface. A protein comprising an internalisation signal istypically constitutively recycled between the endocytic compartment andthe cell surface.

[0062] An internalisation signal typically comprises a 4 or 6 residueinternalisation motif in which the chemical and spatial pattern ofcritical residues is consistent with tight turn structure. Such motifstypically contain an aromatic amino-terminal residue and either anaromatic or large hydrophobic carboxy-terminal residue. Such signals arewell known in the art, for example in Collawn (1991) EMBO J.10,3247-3253and Trowbridge (1991) Current Opinion in Cell Biology, 3, 634-641.

[0063] The intracellular internalisation signal is typically a signalfrom a constitutively recycled receptor. Such receptors include a lowdensity lipoprotein receptor (LDLR), the transferin receptor (TFR), acation-dependent mannose-6-phosophate receptor (CD-Man-6-PR), acation-independent mannose-6-phosophate receptor (CI-Man-6-PR), the polyIg receptor and the asialo glycoprotein receptor (ASGPR).

[0064] The intracellular domain of a protein of the invention maycomprise the entire intracellular domain of a constitutively recyclingreceptor or a fragment thereof. The transmembrane region of a protein ofthe invention may also be derived from a constitutively recyclingreceptor. A fragment of a constitutively recycling receptor typicallycomprises at least one internalisation signal. Preferably the fragmentis from 4 to 40 amino acids in length, for example, from 5, 6, 7, 8, 9or 10 to 20, 25, 30 or 35 amino acids.

[0065] The intracellular domain of a protein of the invention maycomprise a fragment of a first constitutively recycling receptor and afragment of a second constitutively recycling receptor. Such a chimericintracellular domain will typically contain at least one internalisationsignal from each recycling receptor.

[0066] Internalisation of a protein of the invention may be determined,for example, by exposing the surface of a cell expressing the protein toan antibody, virus or other molecule which binds to the extracellulardomain of the protein, incubating the cell with the extracellularantibody or virus, washing to remove surface bound antibody or virus anddetermining the amount of internalised anitbody or virus. Typically,after a 1 hour incubation more than 30%, for example more than 40%, morethan 50% or more than 60% of the protein is internalised. Preferablymore than 70%, for example more than 80% or more than 90% of the proteinis internalised after 1 hour.

[0067] Polynucleotide

[0068] The invention also provides a polynucleotide encoding a proteinof the invention. The polynucleotide may be RNA or DNA. Preferably thepolynucleotide is DNA. The polynucleotide is typically isolated. Apolynucleotide according to the invention has utility in production of aprotein of the invention.

[0069] The present invention also includes expression vectors thatcomprise a polynucleotide encoding a protein of the invention. Suchexpression vectors are routinely constructed in the art of molecularbiology and may, for example, involve the use of plasmid DNA andappropriate initiators, promoters, enhancers and other elements, such asfor example polyadenylation signals which may be necessary, and whichare positioned in the correct orientation, in order to allow for proteinexpression. By way of further example in this regard we refer toSambrook et al, 1989, Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, CSH Laboratory Press.

[0070] Preferably a polynucleotide of the invention in a vector isoperably linked to a control sequence which is capable of providing forthe expression of the coding sequence by the host cell. The term“operably linked” refers to a juxta position wherein the componentsdescribed are in a relationship permitting them to function in theirintended manner. A regulatory sequence, such as a promoter, “operablylinked” to a coding sequence is positioned in such a way that expressionof the coding sequence is achieved under conditions compatible with theregulatory sequence.

[0071] The vectors may be for example, plasmid, virus or phage vectorsprovided with an origin of replication, optionally a promoter for theexpression of the said polynucleotide and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example an antibiotic resistance gene in the case of bacterialplasmid. Vectors may be used in vitro; for example for the production ofDNA or RNA or used to transfect or transform a host cell, for example,in a mammalian host cell.

[0072] Promoters and other expression regulation signals may be selectedto be compatible with the host cell for which expression is designed.Preferably the host cell is a mammalian cell. Mammalian promoters, suchas β-actin promoters, may be used. Tissue-specific promoters arepreferred. Viral promoters, which are readily available in the art, mayalso be used. For example, the Moloney Murine Leukemia Viral LongTerminal repeat (MMLV LTR), the Rous Sarcoma Virus (RSV) LTR promoter,the SV40 promoter, the human cytomegalovirus (CMV) IE promoter and HSVpromoters.

[0073] Cells

[0074] The invention also include cells that have been modified toexpress a protein of the invention. The cells are typically provided invitro. A culture of cells may be provided. Such cells are preferablymammalian cells, such as mouse cells, human cells or other primatecells. Particular examples of cells which may be modified by insertionof vectors encoding for a polypeptide of the invention into mammalianHEK293, HEK293T, CHO, Heta, BHK, 323, COS, Huh7, HepG2 and U937 cells. Acell line may be transiently transfected or is preferably stablytransfected. Generally, the cell line will allow for cell surfaceexpression of the protein.

[0075] A cell expressing a protein of the invention is typicallyobtained by transfecting a cell with a vector of the invention andmaintaining the cell under conditions suitable for obtaining expressionof the protein.

[0076] The invention also includes a cell expressing a protein of theinvention which cell is bound to or infected with the virus to which theextracellular domain of a protein of the invention expressed in saidcell is capable of binding. A cell expressing the protein of theinvention may be infected with a virus by contacting the cell with avirus under conditions suitable for binding of the virus to the proteinof the invention. The viral infection of the cell may be detected by anysuitable means. For example, following viral exposure the virus bound onthe surface of a cell may be removed by acid wash and the cells may bepermeabilised and stained with an antibody to the virus to detect anyviruses that have been internalised into the cell.

[0077] Transgenic Animals

[0078] A protein of the invention may be expressed in cells of atransgenic non-human animal. The transgenic non-human animal istypically of a species commonly used in biomedical research and ispreferably a laboratory strain. Suitable animals include non-humanprimates and rodents. It is preferred that an animal of the invention isa rodent, particularly a mouse, rat, guinea pig, ferret, gerbil orhamster. Most preferably an animal of the invention is a mouse.

[0079] A non-human transgenic animal of the invention may be generatedusing any appropriate protocol. A suitable method comprises:

[0080] (i) making a suitable cell of the invention;

[0081] (ii) allowing the cell to develop into an animal of theinvention; and

[0082] (iii) optionally, breeding the animal true.

[0083] An expression vector of the invention may be introduced into anon-human animal embryo by micro-injection. Or alternatively, embryonicstem cells may be used. Whichever approach is taken, transgenic animalsare then generated. The founder animals that are obtained can be bred.The pro-nuclear microinjection method is preferred, in which a vector ofthe invention is injected into the pronucleus of a fertilized ocyte of anon-human animal.

[0084] A transgenic animal can be analysed for the presence of apolynucleotide of the invention (transgene) in any suitable fashion.Typically, genetic DNA from an animal is screened using PCR primers inthe polynucleotide coding sequence. The results are typically confirmedby blotting the genetic DNA from the positive samples and probing with aradio-labeled polynucleotide of the invention. Expression of a proteinof the invention in cells of a transgenic animal can be determined usingimmunoblotting techniques for example using an anti-F(ab′) antibody oran antibody to the extracellular domain of a cell surface receptor whichcomprises the extracellular domain of a protein of the invention.

[0085] A transgenic non-human animal of the invention may be infectedwith a virus, which virus is capable of binding to the extracellulardomain of a protein of the invention which is expressed in a cell of thetransgenic animal. A transgenic animal may be infected with a virus byexposure to a viral sample by any suitable method.

[0086] A typical viral sample may comprise infectious sera prepared fromanother infected animal, including a human, an infectious tissue culturepreparation such as infected cells or sup ematent from an in vitroculture or an infectious RNA preparation. Typically a viral sample isadministered directly to the transgenic animal. For example, the animalmay be infected by intravenous injection of the virus. The virus may beadministered at any appropriate dosage. A typical dose may be 0.1 ml ofviral infection serum, preferably 0.2 to 0.5 ml.

[0087] A transgenic animal of the invention may express a protein of theinvention in a specific tissue or cell type such that a specific tissueor cell type is infected with the virus. Tissue- or cell-specificexpression of a protein of the invention is achieved using an expressionvector in which a polypeptide of the invention is operably linked to atissue-specific promoter. For example, HCV infects human liver cells. Itis therefore preferred that a transgenic animal expressing a protein ofthe invention capable of binding to HCV expresses the protein in aliver-specific manner. Similarly, it is preferred that a protein of theinvention capable of binding HBV is expressed in liver cells, that aprotein of the invention capable of binding HIV is expressed in T-cellsand/or brain cells, that a protein of the invention capable of bindingrabies virus is expressed at neuromuscular junctions, that a protein ofthe invention capable of binding reovirus type 3 is expressed in neuronsand that a protein of the invention capable of binding Rotavirus isexpressed in gut epithelium.

[0088] Screening Methods

[0089] Cells and transgenic animals according to the invention may beused in methods in screening and testing potential anti-viral agents. Ananti-agent is capable of inhibiting viral entry into a cell, survival ofa virus, viral protein synthesis, viral replication or spread of avirus. Thus, an anti-viral agent may be used as a vaccine to preventviral infection or as a therapeutic agent to treat a viral infection.

[0090] A method for identifying an anti-viral agent, typicallycomprises:

[0091] (i) providing a cell expressing a protein of the invention or anon-human human transgenic animal expressing a protein of the invention;

[0092] (ii) infecting the cell or animal with a virus;

[0093] (iii) contacting the cell or animal with a test agent; and

[0094] (iv) monitoring viral infection;

[0095] thereby determining whether the test agent has anti-viralactivity.

[0096] A method for identifying an anti-viral agent suitable forpreventing a viral infection typically comprises:

[0097] (i) providing a cell expressing a protein of the invention or anon-human transgenic animal expressing a protein of the invention;

[0098] (ii) contacting the animal or cell with a test agent;

[0099] (iii) contacting the cell or animal with a virus capable ofbinding to said protein; and

[0100] (iv) monitoring for infection;

[0101] thereby determining whether the test agent prevents or limitsviral infection.

[0102] Where the method utilises a non-human transgenic animal of theinvention, the test agent and/or the virus may be administered to thesaid animal by any suitable method. Examples of suitable methods ofadministration are included herein.

[0103] Cells expressing a protein of the invention may be contacted invitro with a test agent by any suitable method. The cells may beperfused with the test agent or the agent may be added to the culturemedium bathing the cells. The test agent may be introduced into thecells directly. Any suitable technique known in the art may be used tointroduce the test agent into the cultured cells. Such well-knowntechniques include microinjection, electroporation and methods involvingthe use of transfection agents such as lipofectants, DEAE-dextran andcalcium phosphate.

[0104] Viral infection may be monitored by any suitable method. Forexample, death of host cells, viral replication, protein synthesis orthe presence of viral protein on the surface of host cells may bemonitored. Suitable methods for monitoring viral infection are describedin Chesebro and Wehrly (1988), J. Virol. 62,3779-3788 and in Pincus etal (1989), J. Immunol. 142, 3070-3075.

[0105] HIV infection may be monitored using commercially available kits.For example, an HIV-I p24 ELISA (Coulter Inc, R&D Systems Inc.) or aRT-RCR kit for HIV long terminal repeat (LTR): NASBA [nucleic acidsequence-based amplification] (Amplicar (Roche Diagnostics)) may beused. Suitable assays are also described in the following documents:Steiger et al. (1991), J. Virol. Methods 34(2): 149-160, Byrne et al.(1998), Nucleic Acids Res. 16(9): 4165, Vandamme et al. (1995), J.Virol. Methods 52(1-2): 121-132 and Bolton et al. (1987), J. Clin.Microbiol. 25(8): 1411-1415.

[0106] HCV infection may be monitored using commercially available kitsfor the quantitative (Chiron bDNA signal amplification method) orqualitative (Cobas amplicor, Roche Diagnostics) RT-PCR for the 5′ noncoding region. Use of suitable assays are also described in Lunel et al.(1999), Hepatology 29(2): 528-535, Yeh et al. (1997), J. Virol. Methods65(2): 219-226 and Jacob et al. (1997), Am. J. Clin. Pathol. 107 (3):362-367.

[0107] HBV infection may be monitored by quantitative PCR (Chiron bDNAsignal amplification method; Cobras amplicor (Roche Diagnostics); DigeneDiagnostics, Inc. (DNA: RNA hybridisation)). Suitable assays aredescribed in Khakoo et al. (1996), J. Med. Virol. 50(2) 112-112-116 andChen et al. (1995), J. Virol. Methods 53(1):131-137.

[0108] Test substances may be used at a concentration of from 1 nM to1000 μM, preferably from 1 μM to 10 μM, more preferably from 1 μM to 10μM. A test substance which has anti-viral activity may reduce viralinfection by more than 50%, 60%, 70%, 80%, 90% compared to viralinfection in a control animal or cell.

[0109] Suitable test substances include combinatorial libraries, definedchemical entities and compounds, peptide and peptide mimetics,oligonucleotides and natural product libraries, such as display (e.g.phage display libraries) and antibody products.

[0110] Typically, organic molecules will be screened, preferably smallorganic molecules which have a molecular weight of from 50 to 2500daltons. Candidate products can be biomolecules including, saccharides,fatty acids, steroids, purines, pyrimidines, derivatives, structuralanalogs or combinations thereof. Candidate agents are obtained from awide variety of sources including libraries of synthetic or naturalcompounds. Known pharmacological agents may be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs.

[0111] Therapeutic Uses

[0112] An agent identified by a method of the invention may be used in amethod of therapeutic or prophylactic treatment of the human or animalbody by therapy. The invention provides a method for treating a patientinfected with a virus, the method comprising administering to thepatient a therapeutically effective amount of an agent identified by amethod of determining anti-viral activity of the agent according to theinvention. The patient is generally infected with a virus against whichthe anti-viral agent has been shown to have anti-viral activity. Thepatient may be infected with HCV, HIV, HSV-1, HSV-2, Influenza A,Influenza B, RSV, Rhinovirus, Coxsackie virus or HBV. Preferably thepatient infected with a viral infection is suffering from symptoms ofthe viral infection. For example, a patient infected with HCV ispreferably suffering from hepatitis C.

[0113] The invention provides a method for treating a subject at risk ofviral infection, the method comprising administering to the said subjecta prophylactically effective amount of an agent identified by a methodof the invention. The invention also provides a method for the treatmentof a patient infected with a virus but not suffering from symptoms of adisease caused by the virus in order to prevent the patient developingsaid disease. This method comprises administering an amount of an agentwhich is effective in preventing onset of disease symptoms to a patientinfected with the virus.

[0114] It is preferred that therapeutic treatment is administered in theearly stages of infection. Complications of viral infection, such ascirrhosis, portal hypertension, hepatocellular carcinoma which arecomplications of hepatitis C may also be treated using an agentidentified by a method of the invention.

[0115] An agent for use in a method of treatment of a viral infection bytherapy will typically improve the condition of a patient suffering fromthe infection and/or ameliorate the symptoms of the infection.

[0116] An agent for use in a method of prophylatic treatment of a viralinfection or disease caused by a viral infection will typically lessenthe severity of one or more of the symptoms resulting from infectionand/or may prevent the onset of one or more symptom of infection.

[0117] An agent identified according to a screening method outlinedabove may be formulated with standard pharmaceutically acceptablecarriers and/or excipients as is routine in the pharmaceutical art, andas fully described in Remington's Pharmaceutical Sciences, MackPublishing Company, Eastern Pennsylvania 17^(th) Ed. 1985, thedisclosure of which is included herein of its entirety by way ofreference. Compositions and medicaments for use in a method of treatinga viral infection may be formulated in dosage form. Medicamentscomprising a therapeutic agent identified by a method of the inventionmay be in a form suitable for administration to a patient, for examplein tablet, capsule or liquid form, or may be in a concentrated formsuitable for preparation by a pharmacist.

[0118] The agents may be administered by external or parental routessuch as via oral, buccal, anal, pulmonary, nasal, vaginal, intravenous,intra-arterial, intrahepatic, intramuscular, intraperitoneal,subcutaneous or other appropriate administration routes.

[0119] A therapeutically effective amount of an agent is administered toa patient. An amount of an agent sufficient for preventing infection isadministered to a subject at risk of viral infection. The dose of atherapeutic agent may be determined according to various parameters,especially according to the substance used; the age, weight andcondition of the patient to be treated; the route of administration; andthe required regimen. A physician will be able to determine the requiredroute of administration and dosage for any particular patient. A typicaldaily dose is from about 0.1 to 50 mg per kg of body weight, accordingto the activity of the specific agent, the age, weight and conditions ofthe subject to be treated, the type and severity of the disease and thefrequency and route of administration. Preferably, daily dosage levelsare from 5 mg to 2 g.

[0120] An agent that is capable of preventing viral infection bystimulating the immune system to produce antibodies specific forproteins of the virus is preferably administered in a single dose. Oneor more further doses may be required for long term protection againsthepatitis infection. Further doses may be administered after a period of1 to 15 years after the initial dose, for example after 1, 2, 3, 4, 5,8, 10, 12 or 15 years. Regular doses may be administered at regularintervals after the first dose, for example at 3, 5, 8, 10 or 15 yearlyintervals.

[0121] An agent that is capable of preventing viral infection bystimulating the immune system of a mammal to produce antibodies specificfor proteins of the virus is preferably a nucleic acid. Nucleic acid,such as RNA or DNA, and preferably, DNA, is provided in the form of avector which may be expressed in the cells of the mammal.

[0122] Such nucleic acids may be administered to the animal by anyavailable technique. For example, the nucleic acid may be introduced byinjection, preferably intradermally, subcutaneously or intramuscularly.Alternatively, the nucleic acid may be delivered directly across theskin using a nucleic acid delivery device such as particle-mediated genedelivery. The nucleic acid may be administered topically to the skin, orto the mucosal surfaces for example by intranasal, oral, intravaginal,intrarectal administration.

[0123] Uptake of nucleic acid constructs may be enhanced by severalknown transfection techniques, for example those including the use oftransfection agents. Examples of these agents includes cationic agents,for example, calcium phosphate and DEAE-Dextran and lipofectants, forexample, lipofectam and transfectam. The dosage of the nucleic acid tobe administered can be altered. Typically the nucleic acid isadministered in the range of 1 pg to 1 mg, preferably to 1 pg to 10 μgnucleic acid for particle mediated gene delivery and 10 μg to 1 mg forother routes.

[0124] The following Examples illustrate the invention.

[0125] Materials and Methods

[0126] Cell Lines

[0127] The human embryonic kidney cell line, 293, and its derivative,293T, bearing the large T antigen from SV40 were purchased from AmericanType Cell Collection (Manassas, Va., USA). Huh7 cells were obtained fromJapan Health Sciences Foundation (Chou-ku, Osaka, Japan). HepG2 and U937cells were also purchased from American Type Cell Collection. All cellswere cultured at 37° C. in 5% CO₂ in EMEM, DME or MEM containing 2 mML-glutamine, 1.5 g/L sodium bicarbonate, 0.1 mM non-essential aminoacids, 1 mM sodium pyruvate, and 10% fetal bovine serum.

EXAMPLE 1 Generation of Wild-Type CD81, Chimeric CD81 and Truncated HCVE2(aa384-661) Expression Constructs

[0128] cDNA of the human full length wild-type CD81 (CD81WT) wasobtained by RT-PCR (primers C1 and C2, Table 1) from total cellular RNAof U937 cells and cloned into BamHI/EcOR1 sites of pcDNA3.1+(Invitrogen,Carlsbad, Calif., USA). For CD81-transferrin receptor chimera(CD81-ΔTFR), the N-terminal cytoplasmic domain was obtained by PCR fromthe full-length transferrin receptor (primers C3 and C4, Table 1) andligated to the N-terminal of pcDNA3-CD81WT (Hind III, BamHI). ForCD81-LDLR chimera (CD81-ΔLDLR); the C-terminal cytoplasmic domain ofLDLR was obtained by RT-PCR using total cellular RNA of HepG2 cells (C5and C6, Table 1). PCR product was then ligated to the C-terminal of apcDNA3-CD81WT clone whose stop codon has been removed by PCR method(EcORI, NotI). A schematic representation of the different constructs isshown in FIG. 1A.

[0129] The sequence of HCV E2 cDNA from aa 384-661 was PCR amplifiedfrom the HCV genome of HCV-S1 of genotype 1b (Lim et al. Virus Genes 23,89-95, 2001) and cloned into the BamHI and EcOR5 sites of pSecTagC fromInvitrogen. Primers used are C7 and C8 (Table 1). TABLE 1 Primers usedfor cloning of constructs and detection of viral transcripts. C15′-AAAGCTAGCGGATCCGCCACCATGGGAGTGGAGGGCTGCAC CAAGTGCATCAAGT-3′ C25′-AAAGAATTCGCGGCCGCTCAGTACACGGAGCTGTTCCGGAT GCCACAGCACAGCACCATGCTCAG-3′C3 5′-CCCAAGCTTACCATGGCGATGATGGATCAAGCTAGATCA GCA-3′ C45′-CGCGGATCCCCTTTTTGGTTTTGTGACATTGGC-3′ C55′-CCGGAATTCAAGAACTGGCGGCTTAAGAACATC-3′ C65′-ATAAGAATGCGGCCGCTCACGCCACGT CATCCTCCAG-3′ C75′-GGGGGATCCACCACACCCAAGTGATGGGGG-3′ C85′-GGGGATATCTCTCTGATCTATCCCTGTCCTC-3′ P15′-ACTCATTCCCATTCTGCAGCTTCC-3′ (nt 10-28) P25′-CTGTGAGGAACTACTGTCT-3′ (nt 36-55) P3 5′-CGGTGTACTCACCGGTTCC-3′ (nt161-143) P4 5′-ACTCGCAAGCACCCTATCA-3′ (nt 303-285) P55′-TCGCGACCCAACACTACTC-3′ (nt 274-256) P1TAG5′-TCATGGTGGCGAATAAACTCATTCCCATTCTGCAGCTT CC-3′ TAG5′-TCATGGTGGCGAATAA-3′ HCV 5′-GCAGAAAGCGTCTAGCCATGGCGTTAGTAT-3′ probe:(nt 68-97)

EXAMPLE 2 High Level of CD81 Expressions on the Surface of Stable Huh7Clones

[0130] Transient transfection experiments were performed usingEffectene™ transfection reagent from QIAGEN (Valencia, Calif., USA),according to the manufacturer's protocol. Stable CD81 clones expressingwild-type CD81 (CD81WT), CD81 fused at the N-terminal with 61amino-acids of the cytoplasmic domain of transferrin receptor(CD81-ΔTFR), and CD81 fused at the C-terminal with 50 amino-acids of thecytoplasmic domain of LDL receptor (CD81-ΔLDLR) were generated byelectroporation of 20 μg DNA into about 5×10⁶ Huh7 cells at 0.25 kvoltsusing a BIORAD (Hercules, Calif., USA) gene pulser machine. Cells wereselected by growing in 1 mg/ml of geneticin (GibcoBRL, Gaithesburg, Md.,USA) and single colonies were isolated and analyzed for surfaceexpression by FACScan analysis using antibodies specific for CD81, LDLand transferrin receptors. Stable Huh7 clones were harvested, washed inPBS and resuspended at 1×10⁶ cells/ml. 0.5 ml of cells were incubatedwith a mouse anti-human CD81 antibody or an isotype-matched control fromBD PharMingen (San Diego, Calif., USA) for 30 min at 4° C., washed andre-incubated with a goat anti-mouse antibody conjugated withphycoerythrin (Sigma, St. Louis, USA). Cells were washed and analyzed ona Becton Dickinson flow cytometer (San Jose, Calif., USA). Live cellswere gated and a total of 10 000 events were collected per analysis. Forstable Huh7 cells expressing wild-type CD81, high-expressing clones wereisolated by FACS-sorting. Untransfected parental Huh7 expressed lowlevels of CD81 receptors whereas the 3 stable clones (CD81WT, CD81-ΔTFRand CD81-ΔLDLR) all showed high levels of expression (FIG. 1B). Theexpression levels of endogenous transferrin and LDL receptors in thethree stable clones did not vary significantly from the levels of thesereceptors in untransfected Huh7 cells. 293T cells expressing endogenousCD81, LDL and transferrin receptors were used as positive controls.FACScan analysis for transferrin and LDL receptors was performed usingantibodies from BD PharMingen and Oncogene Research Products (Cambridge,Mass., USA) respectively.

EXAMPLE 3 Chimeric Receptors, CD81-ΔTFR and CD81-ΔLDLR, Internalize MoreEfficiently than CD81

[0131] 50×10⁵ cells were plated on 6-well plates and allowed to settleovernight. 0.5 ml of 10 μg/ml of CD81 monoclonal antibody (Santa CruzBiotechnology, Santa Cruz, Calif., USA) was overlaid onto the cells for1 h at 37° C. After cooling on ice for 10 min, any unbound antibody wasremoved with 3 washes of cold PBS and surface-bound antibody wasstripped by incubating the cells with cold 0.2 M acetic acid/0.5 M NaClfor 5 min, followed by PBS washes. Cells were harvested and lysed inLaemmli's SDS buffer and subjected to western analysis. Briefly, totalprotein was separated on a 10% SDS-polyacrylamide gel and transferred toa nitrocellulose membrane. Then, the membrane was blocked with 5%non-fat dry milk and incubated with goat anti-mouse horse-radishperoxidase (HRP) conjugated antibody (Pierce, Rockford, Ill., USA) for 1h, followed by detection using an enhanced chemiluminescence method(Pierce).

[0132] The heavy chain of the antibody (both surface-bound andinternalized) was observed in CD81WT, CD81-ΔTFR and CD81-ΔLDLR cells(after washes with PBS to remove any unbound antibody from the surface)FIG. 2). As expected, little binding of anti-CD81 antibody tountransfected Huh7 cells was observed. When an additional 5 minute acidwash was used to remove the surface bound CD81 antibody, ˜30% ofanti-CD81 antibody (total amount of surface-bound and internalizedantibodies was normalized to 100%) remained in the intracellularfractions of Huh7-CD81WT cells (FIG. 2). Exposure to antibody for morethan 1 h also did not result in a significant increase in intracellularaccumulation of anti-CD81 antibody in Huh7-CD81WT cells.

[0133] In Huh7-CD81-ΔTFR and Huh7-CD81-ΔLDLR, a higher percentage (70%)of anti-CD81 antibody was internalized, showing that the fusion ofcytoplasmic domains from the two recycling receptors, transferringreceptor and LDL receptor, to CD81 have greatly increased theinternalization efficiency of the receptor (FIG. 2). The fusion of thesecytoplasmic domains at the N-terminal (CD81-ΔTFR) or C-terminal(CD81-ΔLDLR) of CD81 appeared to increase the internalization of CD81 tothe same extent.

[0134] To determine the intracellular localization of wild-type andchimeric CD81 receptors, cells were fixed with 3.7% formaldehyde for 10min at room temperature, following by 10 min permeabilization with 0.2%Triton-X 100, 3.0 min blocking with PBS containing 1% purified BSA(Sigma), 2 h incubation with an anti-CD81 monoclonal antibody (SantaCruz) and finally 1 h incubation with a FITC-conjugated goat anti-mouseantibody (Santa Cruz). Slides were mounted and pictures taken on aMRC1024 laser confocal microscope (BIORAD).

[0135] In all three stable cell lines, punctuate staining around thenucleus was observed, indicating intracellular localization of wild-typeand chimeric CD81 receptors to the endosome structures.

[0136] In order to determine the path of the receptors afterinternalization, living cells were overlaid with anti-CD81 antibody for1 h, followed by acid-stripping of surface-bound antibody. The cellswere then permeabilized as before and probed with an FITC-conjugatedanti-mouse antibody for the presence of internalized anti-CD81 antibody.For Huh7-CD81-ΔTFR and —CD81-ΔLDLR cells, strong intracellular stainingwere observed, showing that anti-CD81 antibody was internalized andtransported to the endosomes, which is the compartment where uncouplingbetween ligand and receptors normally occurs. Consistent with the lowerinternalization efficiency of wild-type CD81, Huh7-CD81WT cells showedlittle intracellular staining in this experiment.

[0137] It should be noted that the measurement of intracellular (i.e.non-acid removable) CD81 antibody as a probe for the internalization ofCD81 receptor is only comparative and may be an underestimation as someof the internalized antibody may be degraded. For example, because rapidintracellular degradation of the antibodies is occurring afterinternalization.

EXAMPLE 4 Chimeric Receptors, CD81-ΔTFR and CD81-ΔLDLR, Undergo GreaterCell Surface Re-Cycling Compared to CD81

[0138] The rate of down-modulation and re-expression of both wild-typeand chimeric CD81 on the cell surface of the various stable clones wasdetermined over a period of 8 h by FACScan analyses. Cells wereincubated with 10 μg/ml of anti-CD81 monoclonal antibody for 1 to 8 h,followed by PBS washes, and then FACScan analysis carried out asdescribed above to determine amount of receptors on cell surface. Meanfluorescence of cells was determined for each time point.

[0139] In general both wild-type and the chimeric CD81 moleculesunderwent a 4 h cycling profile (FIG. 3). CD81WT and CD81-ΔTFR bothshowed a similar profile, with maximum internalization and re-expressionboth occurring within a period of 2 h (FIG. 3). Down-modulation ofCD81-ΔLDLR molecules occurred maximally after a 3 h of exposure toantibody, with re-expression taking place within 1 h later (FIG. 3).Nevertheless, both chimeric CD81 molecules were internalized to agreater degree compared to CD81WT, up to 60% and 44% of CD81-ΔTFR and79% and 50% of CD81-ΔLDLR molecules were internalized, after 2 and 6 hrespectively, of incubation at 37° C., compared to only 33% and 13% ofCD81WT (Table 2). In the case of CD81-ΔLDLR molecules, these continuedto be down-modulated, resulting in up to 82% and 83% internalization atthe end of 3 and 6 h respectively of incubation (Table 2). Moreover,re-expression of chimeric CD81 molecules on the surface wassignificantly more pronounced than wild-type CD81. There was an increasein 90% and 150% of surface CD81-ΔTFR and in 325% and 566% of surfaceCD81-ΔLDLR molecules within the periods of 34 h and 7-8 h respectively,of incubation with antibody, compared to only 47% and 33% re-expressionin CD81WT at the same time periods (Table 2). TABLE 2 Geometric meansfluorescence Incubation at 37° (h) CD81-WT CD81-ΔLDLR CD81-ΔTFR 1 0(unstained) 4.42 2.12 3.19 2 0 345.51 284.37 465 3 0.5 425.25 736.66689.68 4 1 441.27 505.68 645.3 5 2 295.69 152.66 284.11 6 3 293.43130.16 370.81 7 4 436.11 553.42 540.88 8 6 379.9 279.62 300.58 9 7 468.394.15 697.91 10 8 506.24 627.11 750.86

EXAMPLE 5 Binding of Soluble form of HCV E2 Protein to Cell Surface ofHuh7 Stable Clones

[0140] A secreted and soluble form of E2, which lacks the C-terminalhydrophobic transmembrane anchor was expressed in 293T cells bytransient transfection with pSecTagC-E2 using Superfect reagent(Qiagen). 3 days post-transfection, culture medium was cleared andsecreted E2 proteins were captured on NTA-nickel sepharose beads(Qiagen). The beads were washed with PBS and E2 was eluted with 1 Mimidazole solution, followed by centrifugal filtration (Millipore,Bedford, Mass., USA) to remove the imidazole. The pSecTag vector(Invitrogen) contains a secretion signal from the V-J2-C region of themouse IgG kappa-chain.

[0141] Virtually all the expressed E2 protein was secreted into thegrowth medium and could be purified with NTA-nickel sepharose beads asit is fused with a C-terminal polyhistidine tag. In the absence ofreducing agent, the purified protein separated on SDS-PAGE to give twobands: a high molecular weight species >111 kDa (˜80%) and a ˜70 kDaspecies (˜20%) (FIG. 4A). The addition of reducing agent resulted in thedissociation of the higher molecular weight species to give only a 70kDa band on SDS-PAGE (FIG. 4A). This suggests that the majority of theE2 purified were in the form of disulphide-linked aggregates (>111 kDa),which could be dissociated by the addition of reducing agent. Themonomeric E2 exhibited a lower mobility (70 kDa) than the molecularweight predicted from the peptide sequence (˜36 kDa), indicating thatthe protein was heavily glycoslyated.

[0142] A mixed population of E2 proteins in PBS with 1% BSA was overlaidonto monolayers of cells for four hours to assess E2 binding to cellsurface receptors. E2 in PBS with 1% BSA was overlay onto cells for 4 h.Cells were washed 3 times with cold PBS, lysed in Laemmli's SDS bufferand bound E2 were determined by western analysis using an anti-c-mycmonoclonal antibody (Santa Cruz) to detect the C-terminal myc-tag fusedto the E2 protein.

[0143]FIG. 4B shows that E2 bound to Huh7-CD81WT, Huh7-CD81-ΔTFR andHuh7-CD81-ΔLDLR, but not to untransfected Huh7 cells. These resultssuggest that E2 can bind to the extracellular domain(s) of CD81, whichis present in all these stable cell-lines. However, the association rateappeared to be quite slow as bound E2 was only detected after 4 hrs.This is likely to be due to the low concentration of monomeric E2expressed in this system as it has been shown previously that onlymonomeric E2, and not aggregated E2 oligomers, is capable of bindingCD81.

EXAMPLE 6 Chimeric receptors, CD81-ΔTFR and CD81-ΔLDLR Mediate EfficientEntry of HCV Virions into Huh7 Cells

[0144] The ability of the stable CD81-Huh7 clones to support HCV entrywas tested by culturing Huh7 cells stably expressing an inducible fulllength HCV genome (clone SH9; Lim et al., 2001) for 5 days, with orwithout tetracycline (tet) (Sigma), after which the culture media wasremoved, spun at 2500 rpm for 5 min, aliquoted and frozen at −80° C. 800μl of culture media was layered onto 2.5×10⁵ Huh7 cells and stableCD8′-Huh7 clones in 60 mm petri dishes. The cells were added with 1.2 mlcomplete media and incubated for 6-8 h at 37° C., after which they werewashed 6 times with PBS, added with 4 ml of fresh media and re-incubatedfor 5 days at 37° C. At the end of the period, the cells were washed 3×with PBS and total cellular RNA was extracted with the Trizol reagentfrom Gibco BRL, according to the manufacturer's protocol, followed bytreatment with 5U of DNaseI (Promega, Madison, Wis., USA) at 37° C. for30 min.

[0145] The presence of viral particles in the culture media from thesecells was determined by RT-PCR after an antibody capture method usingbeads bound with anti-E2 monoclonal antibodies. 5 μg of anti-E2 (H52,kindly provided by Dubuisson, J, Flint et al., J. Virol. 73, 6235-6244,1999) were bound over-night onto. 10 μl packed volume) of protein A/Gbeads (Oncogene Research Products) at 4° C. Beads were washed twice inPBS and incubated with culture media for 2 h at 4° C. After incubation,the beads were washed extensively in PBS before RT-PCR was carried out.For pre-clearing of the culture medium, a similar protocol was performedexcept, after incubation of culture media with antibody, bound beads(anti-E2 or anti-c-myc for control), the beads were spun down at 14 000rpm for 5 min at 4° C. and the culture media removed and layered ontocells.

[0146] Primers for RT-PCR to detect plus- and minus-strand RNA arelisted in Table 1. A 5 μl volume of the RNA was reverse transcribed at42° C. for 1 h using the specific antisense primer and 200 U ofSuperscript II™ (Gibco BRL. Life technologies), followed by heating at100° C. for 1 h, and treatment with 2.5 μg RNase A for 30 min at 37° C.PCR was carried out with Platiniurn Taq polymerase (Gibco BRL. Lifetechnologies). The first PCR reaction was performed with 5 μl oftemplate in a total volume of 50 μl followed by second round of PCR with1 μl of the first PCR reaction. For the detection of plus-strand RNA,PCR was performed with P1 and P4, followed by P2 and P3 or P5. For thedetection of minus-strand RNA, RT was carried out using the taggedprimer, P1-TAG (Lanford et al., Virology 202, 606-614, 1994). The firstround of PCR was carried out with TAG and P4 and the second round withTAG and P3 or P5. PCR conditions are as follows: 95° C. for 3 min,followed by 30 cycles of 95° C. for 20 sec, 60° C. (plus strand) or 49°C. (minus strand) for 20 sec, 72° C. for 30 sec and a final extension72° C. for 8 min. Amplified products were visualized by ethidium bromidestaining in a 3% agarose gel.

[0147] To determine the specificity of the products obtained by PCRamplification, 25 μl of the PCR products were Southern blotted ontoHybond N+ membrane (USB-Amersham). Hybridisation was carried out with a³²P-end-labelled oligonucleotide corresponding to nt 68 to 97 of the5′NCR (HCV probe) in 5 ng labelled DNA per ml hybridisation buffer(6×SSC, 1× Denhardt's solution, 0.05% Na pyrophosphate, and 100 μg/mlsheared salmon sperm DNA). The filters were incubated at 65° C. for14-16 h after which they were washed twice at 65° C. in 1×SSC-0.1% SDSand 0.5×SSC-0.1% SDS. The filters were then air-dried and exposed toautoradiography films at −70° C. for 4-12 h. Quantification of allauthoradiographs was carried out on a BIORAD densitometer.

[0148] Positive RT-PCR products were observed in culture media oftet-treated cells, but not in untreated cells (FIG. 5, lanes 1 and 3).In addition, these products were still observed after treating the beadswith DNaseI and RNaseA after incubation with culture media fromtet-treated cells (FIG. 5, lane 4), indicating that the bands were not aresult of contamination from DNA or RNA in the media.

[0149] Culture media was layered onto either fresh parental orCD81-expressing Huh7 cells for a period of 6-8 h, after which period thecells were washed extensively and re-incubated with fresh media for 5days. Total RNA was subsequently extracted and RT-PCR performed to assayfor the presence of viral transcripts. Little plus strand RNA was foundin parental Huh7 cells 5 days after exposure to infectious media (FIG.6, lane 1). On the other hand, a low amount of viral transcript wasfound in Huh7 cells over-expressing CD81WT (FIG. 6, lane 2). BothHuh7-CD81-ΔLDLR and Huh7-CD81-ΔTFR exhibited increased levels in viraltranscripts (FIG. 6, lanes 3 and 4) and the values were at least 2.6 and6.9 fold higher than that observed with Huh7-CD81WT, after normalizationwith cellular GAPDH (FIG. 6). No viral products were obtained from cellsinfected with media derived from control un-induced cells.

[0150] To ensure that virus replication had indeed taken place in theinfected cells the presence of minus strand was determined using themethod described by Lanford et al. (1994) that utilizes a tagged reverseprimer. Using a combination of the tagged reverse primer and twodifferent forward primers, we found a similar trend in the expression ofminus transcript in the four cell lines (FIG. 6). No product wasobtained with parental Huh7 cells, whilst both Huh7-CD81-ΔLDLR andHuh7-CD81-ΔTFR cells contained more minus transcripts compared toHuh7-CD81WT cells (FIG. 6). They were respectively 2.7-5.8 and 2.8-10.7folds more than the latter after normalization with cellular GAPDH (FIG.6).

[0151] To confirm the authenticity of the results from the aboveinfection studies, blocking experiments were carried out using anti-E2and —CD81 antibodies. First culture media from tet-treated HCV-Huh7cells was pre-incubated with beads coated with anti-E2 or controlanti-c-myc antibodies for 2 h, after which they were layered onto freshHuh7-CD81WT or —CD81-ΔTFR cells. Thereafter the cells were incubatedfurther for 5 days and analyzed as before for the presence of viraltranscripts. Pre-incubation of media with anti-c-myc beads failed toblock infection of both Huh7-CD81WT and —CD81-ΔTFR cells as both plusand minus strand transcripts were seen after RT-PCR (FIGS. 7A and B,lane 3). Pre-incubation with anti-E2 beads completely abolished viralinfection of these cells as no products were obtained (FIGS. 7A and B,lane 4).

[0152] In another blocking experiment, Huh7-CD81WT and —CD81-ΔTFR cellswere pre-incubated with 2.5 mg/ml anti-human or mouse CD81 antibodiesfor 30 min before a 6 to 8 hour incubation with infectious media. As wasexpected, control anti-mouse CD81 antibodies failed to prevent infectionas viral RT-PCR products were observed in both Huh7-CD81WT and—CD81-ΔTFR cells (FIGS. 7A and B, lane 5). Anti-human CD81 antibodiesprevented infection in Huh7-CD81WT but not in Huh7-CD81-ΔTFR cells(compare FIGS. 7A and B, lane 6). The reason for this difference isunknown, but maybe related to the more pronounced recycling of CD81-ΔTFRcompared to CD81-WT (see also FIG. 3)

EXAMPLE 7 Isolation of Anti-gp120 Antibody Heavy (IgH) and Light (IgL)Chains cDNAs from Hydridoma 902

[0153] cDNAs that encode for the immunoglobulin heavy (IgH) and light(IgL) chains of the anti-gp120 hydridoma 902 were obtained by RT-PCRmethods. The hybridoma 902 (catalog number 521) that produces ananti-HIV-1_(LAV)/HTLV-III_(B) gp120 IgG1 monoclonal antibody wasobtained from the NIH AIDS Research & Reference Reagent Program(Chesebro & Wehrly, J. Virol. 62, 3799-3788,1988; Pincus et al., J.Immunol. 142, 3070-3075, 1989). Total RNA was extracted from thehybridoma using a RNeasy kit (Qiagen, Valencia, Calif., USA) andtranscripted into cDNA using SuperscriptII RNase reverse transcriptase(GibcoBRL, Gaithesburg, Md., USA). PCR reaction was then carried outusing the Expand long template PCR system from Roche MolecularBiochemicals (Indianapolis, Ind., USA) and the following primers: IgL:Forward: 5′ ACCATGAAGTTTCCTTCTCAACTTCTGCTCTTCC 3′ Reverse:5′ GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA 3′ IgH: Forward:5′ GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT 3′ and5′ ACTAGTCGACATGGACTCCAGGCTCAATTTAGTTTT CCT 3′ (R = A or G; Y = C or T;S = C or G; W = A or T) Reverse: 5′ TTATTTACCAGGAGAGTGGGAGAGGCTCTT 3′

[0154] PCR products were cloned into pCRII-TOPO vector using the TOPO TAcloning kit (Invitrogen, Carlsbad, Calif., USA) and DNA sequencing ofthe plasmids was carried out in the core facility at the Institute ofMolecular and Cell Biology. 200 ng of the double-stranded templates and10 ng of the primer were used for the dideoxy method with the TaqDyeDeoxy terminator cycle sequencing kit and the automated DNA sequencer373 from PE Applied Biosystems (Foster City, Calif., USA).

[0155] Secreted antibodies from the hybridoma were captured onto proteinA/G beads (Oncogene Research Products, Cambridge, Mass., USA) and thenthe beads extensively washed with PBS, before the bound proteins wereeluted with Laenunli's SDS buffer by heating at 100° C. for 5 minutesand separated on a 15% SDS-polyacrylamide gel. The two major bands at˜55 kDa and ˜20 kDa were excised from the gel and processed for Edmansequencing on a Perkin Elmer machine according to manufacturer'sprotocols.

[0156] N-terminal sequencing of the mature heavy and light chain of theantibody secreted by the hybridoma 902 gave peptide sequences ofEVQLQQSGAE and DIQMTQSSSY respectively. These sequences of the matureantibody matched to amino acid number 18 and 21 onwards respectively ofthe proteins encoded by the cDNAs, thus confirming the correctimmunoglobulin genes have been isolated (FIG. 8).

EXAMPLE 8 Cloning of IgL and IgH into Expression Vectors andConstruction of Chimeric Proteins

[0157] IgL was cloned into Kpn I and Not I sites of pXJ41neo and IgH wascloned into Kpn I and Hind III sites of pCep4 vector (Invitrogen).

[0158] Chimeric heavy chain IgH-ΔCI-MPR was constructed by fusing thetransmembrane domain and the cytoplasmic tail of CI-MPR (aa 2305-2492)to the C-terminal of the full-length IgH clone. Similarly, IgH-ΔLDLR wasconstructed by the addition of the transmembrane region and cytoplasmictail of LDLR (aa 790 to 860). C-terminal 188 aa of the humancation-independent mannose 6-phospate receptor (CI-MPR) and 71 aa of thehuman low density lipoprotein receptor (LDLR) were obtained by RT-PCRusing total cellular RNA from HepG2 cells as described above. Theprimers used were: CI-MPRfor: 5′ CCCAAGCTTGCAGTCGGCGCGGTGCTCAGC 3′CI-MPRrev: 5′ ATAAGAATGCGGCCGCTCAGATGTGTAAGAGGTCCT CGTC 3′ LDLRfor:5′ CCCAAGCTTCTGTCCATTGTCCTCCCCATC 3′ LDLRrev:5′ ATAAGAATGCGGCCGCTCACGCCACGTCATCCTC CAG 3′

[0159] The PCR products were then ligated into the 3′ end of pCep4-IgHusing the Hind III and Not I sites, to give chimeric heavy chains,IgH-ΔCI-MPR and IgH-ΔLDLR respectively.

EXAMPLE 9 Chimeric IgH Associates with IgL and Chimeric RecombinantAntibody Expresses on Cell Surface

[0160] Transient transfection experiments were performed usingEffectene™ transfection reagent from QIAGEN (Valencia, Calif., USA),according to the manufacturer protocol. To generate stable clones thatconstitutively express the IgL chain, 20 μg of DNA were mixed with about5×10⁶ 293 cells and electroporated at 0.25 kvolts using a BIORAD(Hercules, Calif., USA) gene pulser machine. Cells were selected bygrowing in 0.4 mg/ml of geneticin (GibcoBRL) and single colonies wereisolated and total protein analyzed by western analysis. A single clonethat expressed high level of IgL was then re-transfected in the samemanner with various IgH DNA and cells were selected by growing in 0.2mg/ml hygromycin B (Roche). Single colonies were isolated and totalprotein analyzed by western analysis.

[0161] Transfected cells were lysed in HBS buffer (10 mM Hepes, 150 mMNaCl, 1% NP40) and total protein concentration determined by CosmassiePlus reagent from Pierce (Rockford, Ill., USA). 30 ug of total wereseparated on a 15% SDS-polyacrylamide gel and transferred tonitrocellulose membrane. Then, the membrane was blocked with 5% non-fatdry milk. For detection of IgL protein, the blot was incubated overnightwith a horse-radish peroxidase (HRP) conjugated anti-light chainantibody from BD Pharmingen (San Diego, Calif., USA), followed bydetection using an enhanced chemiluminescence method Pierce). For theheavy chain, the blot was incubated with an HRP-conjugated goatanti-mouse (Pierce) antibody before detection.

[0162] Transient expression of each chimeric heavy chain respectivelytogether with the full-length light chain (IgL) showed that most of theheavy and light chains are retained in the cell. In contrast,transfection of the original IgH together with IgL resulted in secretionof the recombinant antibody into cell culture medium (FIG. 9A). Inaddition, there is proper association between the different heavy chainsand light chain in solution (either cell culture medium for IgH or celllysate for IgH-ΔCI-MPR and IgH-ΔLDLR) as capturing the heavy chains withprotein A/G beads (which binds to constant region of IgH) also pulleddown IgL proteins (FIG. 9A).

[0163] Cell surface expression was analysed by FACScan analysis:Transiently transfected 293T cells were harvested, washed in PBS andresuspended at 1×10⁶ cells/ml. 0.5 ml of cells were incubated with agoat anti-F(ab′)₂ antibody (9 ug/ml) conjugated with FITC (Pierce) for30 min at 4° C., washed and analyzed on a Becton Dickinson flowcytometer (San Jose, Calif., USA).

[0164] FACScan analysis showed that IgL complexed with IgH-ΔCI-MPR orIgH-ΔLDLR were expressed on the cell surface so that they can bind ananti-F(ab′)₂ antibody (FITC conjugated) (FIG. 9B). In contrast, cellstransfected with the original IgH and IgL were not able to bind theanti-F(ab′)₂ antibody.

EXAMPLE 10 Cell Surface-Displayed Chimeric Antibodies UndergoInternalization

[0165] Stable clones, 293-IgH-AM6PR and 293-IgH-ΔLDLR, which expresseshigh levels of heavy and light chains, were obtained from transfectionof 293 cells (FIG. 9C). To determine if these clones can bind andinternalize an anti-F(ab′)₂ antibody, live cells were overlaid with theanti-F(ab′)₂ antibody to allow endocytosis to occur, followed by PBSwashes and cell fixation and confocal microscopy as described below.

[0166] 30,000 live cells were plated onto poly-D-lysine (Sigma, StLouis; USA) treated 4-well Permanox slide chambers (Nalge NuncInternational Corp., IL, USA) and allowed to settle overnight. Then 0.2ml of 7.5 μg/ml of goat anti-mouse F(ab′)₂-FITC antibody (Pierce) wasoverlaid onto the cells for 1 h at 37° C. The plate was then cooled onice for 5 min before any unbound antibody was removed with 3 washes ofcold PBS. For stripping of surface-bound antibody, the cells werefurther incubated with cold 0.2 M acetic acid/0.5 M NaCl (Haigler etal., J. Biol. Chem. 255, 1239, 1980) for 5 min, followed by PBS washes.Cells were fixed with 3.7% formaldehyde for 10 min at room temperaturefollowing by PBS washes. Slides were mounted and pictures taken on aMRC1024 laser confocal microscope (BIORAD).

[0167] A large amount of total (surface and intracellular) anti-F(ab′)₂antibodies was observed in 293-IgH-ΔCI-M6PR and 293-IgH-ΔLDLR cells butnot in the parental 293 cells. When an additional acid wash was includedto remove surface bound anti-F(ab′)₂ antibody before cell fixation,internalized anti-F(ab′)₂ antibodies were observed in 293-IgH-ΔCI-MPRand 293-IgH-ΔLDLR cells and were localized in intracellular spot-likestructures. The data showed that the stable clones are expressingproperly assembled chimeric antibodies on cell surface and thesechimeric antibodies can be rapidly internalized.

EXAMPLE 11 Antibody Secreted by Hybridoma and Recombinant Antibody CanBind HIV-1 MC99IIIBΔTat-Rev Virus In Vitro

[0168] The attenuated HIV-1 MC99IIIBΔTat-Rev virus, which can onlypropagated in CEM-TART cells that constitutively express tat and rev,provided a safe source of high titre HIV-1 virus. The HIV-1MC99IIIBΔTat-Rev virus (catalog number 1943) and CEM-TART cells (catalognumber 1944) were obtained from the NIH AIDS Research & ReferenceReagent Program. Viruses were propagated as previously described andstored at −70° C.

[0169] For in vitro binding experiments to confirm that antibodiessecreted by hybridoma 902 can recognise the gp120 envelope protein onHIV-1 MC99IIIBΔTat-Rev virus, secreted antibodies from hybridoma 902were captured onto protein A/G (which binds to IgH constant region),followed by PBS+0.2% NP40 washes and then overnight incubation withHIV-1MC99IIIBΔTat-Rev virus diluted 10 times with the same buffer. Thebeads were washed and any bound virus eluted by boiling the beads inLaemmli's SDS buffer and detected by western analysis using ananti-gp120 antibody (NEN Life Science Products Inc., Boston, Mass.,USA). Antibody secreted into the culture medium following transienttransfection of 293T was tested for virus binding in the same manner.

[0170] As shown in FIG. 10, beads which adsorbed antibodies secreted bythe hybridoma or antibodies secreted by 293T cells, that weretransiently transfected with IgL and IgH, were capable of binding virus.No binding of virus was observed when the cells were transfected withIgH only.

EXAMPLE 12 Cells Expressing 293-IgH-ΔCI-MPR and 293-IgH-ΔLDLR Bind andInternalize HIV-1 MC99IIIBΔTat-Rev Virus

[0171] Overlay experiment were performed as described above foranti-F(ab′)₂ antibody except that the virus was overlaid was for 1 and 3hours followed by acid wash to remove surface-bound virus and cellfixation. Then, the cells were permeabilized for 10 minutes with 0.2%Triton-X 100, blocked for 30 min with PBS containing 1% purified BSA(Sigma), before incubation for 2 h with an anti-HIV-p24 monoclonalantibody (NEN Life Science Products Inc.), followed by incubation for 1h with a FITC-conjugated goat anti-mouse antibody (Santa CruzBiotechnology, Santa Cruz, Calif., USA).

[0172] Live 293-IgH-AM6PR and 293-IgH-ΔLDLR cells were overlaid withMC99IIIBΔTat-Rev virus as described above for 1 h or 3h at 37° C.,followed by acid wash to remove any surface bound virus. The cells werefixed and permeabilized before staining of any virus inside the cellswas performed with an anti-HIV-p24 antibody. After 1 h, strongintracellular staining was observed in both of 293-IgH-ΔCI-M6PR and293-IgH-ΔLDLR cells but not in the parental cells. The virus appeared tobe have been internalized and transported to Golgi-like intracellularstructures within 1 h. After 3h, even stronger intracellular stainingwas observed and the cells appeared more shrunken, indicating that theinternalized virus may have some cytopathic effects on the cells.

EXAMPLE 13 Cells Expressing Chimeric Antibody IgL/IgH-ΔCI-M6PR can beInfected by a Pseudotype UV Virus

[0173] 293-IgH-ΔCI-MPR (clone 9) and 293-IgH-ΔLDLR (clone 10) stableclones were analyzed for surface expression of chimeric antibodies byFACScan analysis. 293-IgH-ΔCI-MPR cells showed higher surface expressionof antibodies then 293-IgH-ΔLDLR (FIG. 11A). The cells were infectedwith a pseudotype-virus, HXB2 (He and Landau, 1995, J. Virol., 69:4587-4592). This pseudotype virus contained a luciferase gene insertedinto the nef gene and infection of cells with this reporter virus wasmeasured by determining the amount of luciferase gene product present inlysates of the cells using commercial reagents (Promega, USA). For PMAtreatment, at 84h post-infection, the infected cells were treated with100 nM PMA for 18h before the cells were lysed and assayed forluciferase activity. 293-IgH-ΔCI-MPR showed luciferase activities (˜9fold greater than in untreated cells) only in infected cells that weretreated with PMA and not in infected cells that were not treated withPMA (FIG. 11B). This indicates that 293-IgH-ΔCI-MPR cells allow thereplication of internalised virus. 293-IgH-ΔLDLR cells did not showsignificant luciferase activities even after PMA treatment and this maybe due to the lower expression of chimeric antibodies on the surface ofthese cells in comparison to 293-IgH-ΔCI-MPR (see FIG. 11A).

EXAMPLE 14

[0174] Materials and Methods

[0175] Sera from HCV-Infected Patients

[0176] Serum samples were obtained from two patients with HCV infectionbeing followed at the National University Hospital, Singapore. Theamount of HCV RNA in these two sera were quantified by the branched DNAmethod (Quantiplex HCV-RNA assay; Chiron Corp. (Emeiyville, Calif.,USA)) at the Molecular Diagnosis Centre, National University Hospital,Singapore, and any remaining samples were stored at −80° C. The viralloads of the serum of patient A and B were 63.4×10⁶ equivalents/ml and>120×10⁶ equivalents/ml, respectively. After it was confirmed that nofurther testing of these samples were necessary, they were transferred,with the administrating doctor's consent, to our laboratories for theexperiments.

[0177] Internalization of HCV Particles

[0178] 5×10⁵ cells were plated on 6 cm plates and allowed to settleovernight. The cells (about 50% confluent) were washed twice with DMEMmedium without FBS, followed by addition of diluted serum (100 μl ofserum with 900 μl of DMEM medium without FBS), untreated or treated withdetergent or pre-cleared with antibodies as described below. The cellswere left for at least 8 h in a 37° C. incubator, 5% CO₂, before theywere washed extensively with DMEM without FBS. Finally, 2 ml of completeDMEM medium was added to the cells and the cells were returned to theincubator. Two days later, the cells were trypsinized and all the cellstransferred to a 15 cm plate for further incubation. Another 5 dayslater, the cells were trypsinized and collected and washed twice withDEPC-treated (Sigma) PBS and used immediately or stored at −80° C.

[0179] For treatment with detergent, sera were diluted 10 times withDMEM medium without FBS and sterile-filtered deoxychloate solution(Sigma, 0.5% stock) was added to a final concentration of 0.05% andmixed at 4° C. for 4 h. Bio-beads (BIORAD) were prepared as described inmanufacturer's protocol (using DEPC-treated PBS) and 2 ml bead slurrywas added to the detergent-treated serum and incubated overnight at 4°C. This step removed the detergent from the serum to prevent any toxiceffects on the cells. Then the beads were allowed to settle by gravityand the supernatant carefully remove and overlaid on the cells.

[0180] For the pre-clearing experiments, 1 ml of diluted serum (100 μlof serum with 900 μl of DMEM without FBS) was incubated with 10 μg ofeither anti-HCV E2 antibody (Austral Biologicals) or anti-c-myc antibody(Santa Cruz) and 100 μl of protein A/G beads (Oncogene ResearchProducts) overnight at 4° C. After that, the beads were spun down andthe supernatant overlaid on the cells. The beads were washed twice withDEPC-treated PBS and total RNA was extracted from the immuno-complexeswith Trizol LS (Gibco BRL), according to the manufacturer's protocol,and resuspended in 30 μl of DEPC-treated water. Each RNA sample (neat,10×, 30× and 100× dilutions) was tested for positive strand HCV RNA asdescribed below.

[0181] RNA Extraction, Reverse Transcription and Nested PCR

[0182] Total RNA from ˜3×10⁶ cells were extracted with Trizol (GibcoBRL) according to the manufacturer's protocol, and resuspended in 60 μlof DEPC-treated water. 5 μl of RNA was treated with 5 U of RNase-freeDNaseI (Roche Diagnostics, GmbH, Mannheim, Germany) at 37° C. for 1 h,followed by inactivation of DNaseI by heating at 100° C. for 10 min. Forthe detection of positive strand HCV RNA, 5 μl of DNaseI-treated RNA wasthen reverse transcribed at 42° C. for 1 h using the antisense primer209 (Table 3), dNTP (Roche) and 100 U of Superscript II RNase H reversetranscriptase (Gibco BRL) in a final volume of 10 μl, followed byinactivation by heating at 70° C. for 15 min. This product was used astemplate for nested PCR. The first PCR reaction was carried out usingthe external primers (antisense primer 209 and sense primer 939, Table3) with 5 μl template in a total volume of 25 μl and the second round ofPCR then performed using internal primer set (antisense primer 211 andsense primer 940, Table 3) with 1 μl of the first PCR reaction astemplate.

[0183] For the detection of negative strand HCV RNA, the tagged primermethod devised by Lanford et al., Virology 202, 606-614, 1994, was used.5 μl of DNaseI-treated RNA was reverse transcribed as above except thatthe tagged primer, TAGNC1 (sense, Table 3) was used instead. Afterinactivation of the reverse transcriptase, the template was furthertreated with 0.1 mg/ml of RNaseA (Sigma) at 37° C. for 30 min to degradeany remaining RNA. Then, 5 μl of the template was used for the firstround of PCR using external primer set, TAG and antisense 209 (Table 3).This was followed by a second round of PCR using internal primer set,TAG and antisense 211 (Table 3), using 1 μl of the products from thefirst PCR as template.

[0184] Cellular GAPDH mRNA was used to check that similar number ofcells was used in each experiment. For this purpose, 1 μl ofDNaseI-treated RNA was then reverse transcribed as above except thatoligodT (Roche) was used instead. Then 1 μl of the template was used forone round of PCR using the primers, GAPDHfor and GAPDHrev (Table 3).

[0185] All PCR were performed using Titanium Taq DNA polymerase fromClonetech Laboratories Inc. (Palo Alto, Calif., USA) in a final volumeof 25 μl and PCR conditions are as follows: 95° C. for 2 min, followedby 35 cycles of 95° C. for 30 sec, 55° C. for 30 sec, 72° C. for 1 min,and a final extension at 72° C. for 10 min. Amplified products (25 μl)from the second round of PCR for positive and negative strand viraltranscripts, or from one round of PCR for GAPDH, were separated on a 3%agarose gel and visualized under UV light after staining with ethidiumbromide.

[0186] Southern Hybridization

[0187] For Southern blotting, the negative strand RT-PCR products wereseparated on agarose gel and blotted onto Hybond-N membrane(Amersham-Pharmacia Biotech). Hybridization was carried out with anoligonucleotide, corresponding to sequence in the 5′NCR(HCV probe, Table1), 5′-labeled with [γ-³²P]dATP (NEN Life Science Products, Boston,Mass., USA) by T4 polynucleotide linase (New England Biolabs).ExpressHyb hybridization solution (Clonetech) and 10 μg/ml of shearedsalmon sperm DNA (Sigma) were used for the hybridization. The filterswere incubated at 60° C. for 14-16 h after which they were washed twicewith 2×SSC-0.1% SDS and twice with 1×SSC-0.1% SDS, at room temperatureand finally twice in 0.1×SSC-0.1% SDS, at 60° C. The filters werewrapped in SaranWrap and exposed to autoradiography films at −80° C. for12-24 h.

[0188] TA Cloning and Sequence Analysis

[0189] After separation on agarose gel, positive strand RT-PCR productswere excised and purified using the QLAquick kit (Qiagen) and ligatedinto pCRII-TOPO vector using the TA cloning kit from Invitrogen. Atleast 3 clones from each ligation were sequenced (with both M15 forwardand reverse primers) and confirmed to be identical. Sequences werealigned using MegAlign software (DNAStar Inc., Madison, Wis., USA).

[0190] DNA sequencing of all constructs was carried out by the corefacility at the Institute of Molecular and Cell Biology. 200 ng of thedouble-stranded templates and 10 ng of the primer were used for thedideoxy method with the Taq DyeDeoxy terminator cycle sequencing kit andthe automated DNA sequencer 373 from PE Applied Biosystems (Foster City,Calif., USA).

[0191] Results

[0192] Chimeric Receptor, TfR-CD81, Mediate Efficient Entry of HCVParticles into Huh7 Cells

[0193] We tested the ability of the stable Huh7 clones to mediate HCVentry. Serum from two HCV-infected patients (A and B) were overlaid ontountransfected Huh7 or Huh7-CD81WT or Huh7-TfR-CD81 cells for a period of˜8 h, after which these 10, cells were washed extensively andre-incubated with fresh media for 7 days. Total RNA was subsequentlyextracted from the cells and nested RT-PCR performed to assay for thepresence of positive strand viral transcripts as an indicator ofinternalization of HCV particles. As the genotypes were initially notknown, the sequences of primers used for RT-PCR were chosen from regionsin 5′ non-coding region (5′ NCR) that are well conserved between allknown HCV variants. These primers (Table 3) correspond to nucleotidepositions (nt) 36 to 73 (sense) and nt 331 to 279 (antisense) ofrepresentative 1b isolate HCV-BK (Takamizawa et al., J. Virol. 65,1105-1113, 1991; GenBank accession number M58335) and are similar to theones commonly used in the literature for genotyping of HCV isolates.

[0194]FIG. 12A shows the results obtained using serum from patient A. Nopositive strand HCV-specific RT-PCR product was detected foruntransfected Huh7 or Huh7-CD81WT cells but a strong signal was observedfor Huh7-TfR-CD81 cells, showing that a significant amount of H1CVparticles was internalized into Huh7-TfR-CD81 cells (FIG. 12A, panel I).When the same experiments were performed with serum from patient B, weinitially found no positive strand RT-PCR products for untransfectedHuh7 or Huh7-CD81WT or Huh7-TfR-CD81 cells (FIG. 12B, panel I, lane1-3). As it has been reported that some virus particles in patient seraare heavily coated with lipoproteins and/or anti-viral antibodies (IgG),we repeated the experiment after treating the patient serum with 0.05%of a mild detergent, deoxychloate. This method was reported to removelipoproteins/IgG gently from virus particles, resulting in envelopedvirions, which can be trapped onto grids by an anti-HCV E2 antibody andvisualized with electron microscopy. Remarkably, after the detergenttreatment, efficient uptake of viral particles was observed forHuh7-TfR-CD81 cells but not for untransfected Huh7 or Huh7-CD81WT cells(FIG. 12B, panel I, lane 4-6).

[0195] The presence of negative strand RNA, which is an indicator ofactive viral replication, was examined using the strand-specific taggedRT-PCR method devised by Lanford et al., 1994. Again, no product wasobtained with untransfected Huh7 and Huh7-CD81WT cells, whereasHuh7-TfR-CD81 cells showed a weak RT-PCR product under UV light afterstaining with ethidium bromide (FIGS. 12A and B, panel III). Furtherconfirmation was obtained by Southern blot analysis (FIGS. 12A and B,panel IV). The low level of negative strand RNA observed may be due to alow level of replication in Huh7 cells or could be that the time-point(7 days after infection) used was not optimal. Analysis of cellscollected at later time-points would be needed to address this, forexample, intermittent detection of negative strand has been reported ininfected primary human hepatocytes.

[0196] RT-PCR products from patient A and B (Huh7-TfR-CD81 cells) wereligated into the pCRII-TOPO vector (Invitrogen) and the sequences of theinserts were determined. Sequence of the RT-PCR product (205 bp) frompatient A is identical to the corresponding 5′ NCR region ofrepresentative genotype 1b, HCV-BK, sequence (Takamizawa et al., 1991;Genbank accession number M58335) and that of patient B shows twomismatches (FIG. 12C). Therefore, both patients A and B are most likelyto be infected with HCV genotype 1b.

[0197] Pre-Clearing of Patient Serum with an Anti-E2 Antibody AbolishedInternalization of HCV Particles into Huh7-TfR-CD81 Cells.

[0198] The remaining serum of patient A was frozen after the overlayexperiment described above. Here, the serum was thawed and used to testif an anti-E2 antibody, which can detect glycoslyated E2 proteins (datanot shown), can immunoprecipate HCV particles from the serum. Afterincubating the serum with anti-HCV E2 antibody or control antibody(anti-c-myc antibody) and protein A/G beads, the beads were washed, RNAextracted, and RT-PCR performed to detect for positive strand HCV RNAassociated with the beads. As shown in FIG. 13A, some viral RNA wasimmunoprecipated by both the anti-HCV E2 and anti-c-myc antibody.However, when we diluted the extracted RNA by 10×, 30× and 100× beforeperforming the RT-PCR, RT-PCR products were observed only for anti-HCVE2 antibody at 10× and 30× dilutions but not for control anti-c-mycantibody, showing that amount of HCV particles immunoprecipated with theanti-HCV E2 antibody was 10 to 30 times higher than with anti-c-mycantibody (FIG. 13A).

[0199] The sera after pre-clearing with anti-HCV E2 or anti-c-mycantibody, were then used for overlaying onto Huh7-TfR-CD81 cells. Thecells were then tested for presence of positive strand HCV RNA asbefore. As shown in FIG. 13B, anti-E2 antibody remove significant amountof viral particles from the serum such that no positive strand RT-PCRwas observed in Huh7-TfR-CD81 cells that were overlaid with the serumpre-cleared with anti-E2 antibody (FIG. 13B, lane 5). In contrast,positive strand RT-PCR product was clearly detected when the serum waspre-cleared with anti-c-myc antibody (FIG. 13B, lane 6).

[0200] Here, we also repeated the overlaying experiments to compare theinternalization efficiency between untransfected Huh7, Huh7-CD81WT,Huh7-TfR-CD81 and Huh7-CD81-LDLR cells. Huh7-CD81-LDLR cells were notused in the first two overlay experiments described in FIGS. 12A and B.The results showed that positive strand RT-PCR products were observed inHuh7-TfR-CD81 and Huh7-CD81-LDLR cells, but not in untransfected Huh7 orHuh7-CD81WT cells (FIG. 13B, lanes 1-4).

[0201] In summary, in 3 independent experiments (using two differentpatient sera), internalization of positive strand HCV RNA wasconsistently observed in Huh7-TfR-CD81 cells but not in untransfectedHuh7 or Huh7-CD81WT cells. And in one experiment, internalization ofpositive strand HCV RNA into Huh7-CD81-LDLR cells was also observed.Pre-clearing of patient serum with an anti-HCV E2 antibody efficientlyremoved HCV particles so that no internalization of viral RNA wasobserved for Huh7-TfR-CD81 cells (FIG. 13B), suggesting that HCV E2protein is the direct ligand for CD81, in agreement with previousstudies. We concluded that by engineering endocytotic signal tags fromtransferrin and LDL receptors into either the N- or C-terminuscytoplasmic domains of CD81, we were able to trigger the uptake of viralparticles in the serum of HCV-infected patients into Huh7 cells. TABLE 3Primers used for the detection of viral transcripts. Name Sequence(5′ to 3′) 209² ATACTCGAGGTGCACGGTCTACGAGACCT (nt 331-312)¹ 939³CTGTGAGGAACTACTGTCTT (nt 36-55)¹ 211² CACTCTCGAGCACCCTATCAGGCAGT (nt294-279)¹ 940³ TTCACGCAGAAAGCGTCTAG (nt 54-73)¹ TAGNC1TCATGGTGGCGAATAAACTCCACCATAGATCACTCC (nt 15-34)¹ TAG TCATGGTGGCGAATAAHCVprobe GCAGAAAGCGTCTAGCCATGGCGTTAGTAT (nt 59-88)¹ GAPDHforCTGAGAACGGGAAGCTTGTCATCA GAPDHrev CGTCTAGCTCAGGGATGACCTTG

1. A chimeric transmembrane protein comprising: (i) an extracellulardomain capable of binding a virus; and (ii) an intracellularinternalisation signal, wherein the chimeric transmembrane protein iscapable of internalizing a virus bound to the extracellular domain.
 2. Aprotein according to claim 1, wherein said extracellular domain is anantibody fragment.
 3. A protein according to claim 2, wherein saidantibody fragment comprises a variable region of an antibody heavy chainand a variable region of an antibody heavy chain.
 4. A protein accordingto claim 1 wherein said extracellular domain comprises a cell-surfacereceptor, or fragment thereof, capable of binding to a viral coatprotein.
 5. A protein according to claim 1 wherein said virus isselected from the group consisting of HCV, HIV, HBV, RSV, influenzavirus, herpes simplex virus, rabies virus, coxsackie virus andrhinovirus.
 6. A protein according to claim 1 wherein said virus is HIV.7. A protein according to claim 6 wherein said extracellular domain isan antibody to gp120/160, or a fragment of said antibody.
 8. A proteinaccording to claim 6 wherein said extracellular domain is CD4, or afragment thereof capable of binding HIV.
 9. A protein according to claim1, wherein said virus is HCV.
 10. A protein according to claim 9 whereinsaid extracellular domain is an antibody to E2 protein or a fragment ofsaid antibody.
 11. A protein according to claim 9 wherein saidextracellular domain is CD81, or a fragment thereof capable of bindingHCV.
 12. A protein according to claim 1 wherein said internalisationsignal is from a constitutively recycling receptor.
 13. A proteinaccording to claim 12 wherein said receptor is selected from the groupconsisting of low density lipoprotein receptor (LDLR), tranferinreceptor (TFR), cation-dependent mannose-6-phosphate receptor(CD-Man-6-PR), cation-independent mannose-6-phosphate receptor (CIMan-6-PR), poly Ig receptor and asialo gylcoprotein receptor (ASGPR) H1subunit.
 14. A protein according to claim 12 wherein said proteincomprises the entire intracellular domain of said receptor.
 15. Aprotein according to claim 12 wherein said protein comprises a fragmentof said receptor.
 16. A protein according to claim 15, wherein saidfragment is from 4 to 40 amino acids long.
 17. A protein according toclaims 12 wherein said protein comprises a chimeric intracellular domaincomprising a fragment of each of two or more constitutively recyclingreceptors.
 18. A polynucleotide encoding a protein according to claims1.
 19. A vector comprising a polynucleotide according to claim
 18. 20. Acell comprising a protein according to any one of claims
 1. 21. A methodof producing a cell comprising the protein of claim 1, said methodcomprising transfecting a cell with a polynucleotide encoding theprotein or a vector comprising a polynucleotide encoding the protein andmaintaining said cell under conditions suitable for obtaining expressionof the protein.
 22. A cell according to claim 20 which is infected witha virus capable of binding to the extracellular domain of said protein.23. A cell according to claim 22 which is a non-human cell.
 24. A cellaccording to claim 22 which cell is a liver cell and wherein said virusis HCV.
 25. A cell according to claim 22, wherein said virus is HIV. 26.A method of producing a cell according to claim 22, which methodcomprises contacting a cell according to claim 20 with a virus underconditions suitable for binding of said virus to said protein.
 27. Atransgenic non-human animal comprising a cell according to claim
 20. 28.A method for identifying an anti-viral agent, said method comprising:(i) providing a cell according to claim 22 or a non-human transgenicanimal comprising a cell according to claim
 22. (ii) contacting saidcell or animal with a test agent; and (iii) monitoring viral infectionthereby determining whether the test agent has anti-viral activity. 29.A method of identifying an anti-viral vaccine or agent capable ofpreventing or inhibiting viral infection, which method comprises: (i)providing a cell according to claim 20 or a non-human transgenic animalcomprising a cell according to claim 20; (ii) contacting said cell oranimal with a test agent; (iii) contacting said cell or animal with avirus capable of binding to said protein; and (iv) determining whetherthe test agent prevents or limits viral infection.
 30. A methodaccording to claim 28 wherein in step (ii) said test agent isadministered to said animal.
 31. A method according to claim 28 wherein(iii) comprises one or more of the following: (a) monitoring cell death;(b) monitoring viral replication; (c) monitoring protein synthesis; and(d) monitoring presence of viral protein on the surface of said cells.32. An anti-viral agent identified by a method according to claim 28.33. Use of an anti-viral agent according to claim 32 in the manufactureof a medicament for use in the therapeutic or prophylactic treatment ofa viral infection.
 34. Use according to claim 31 wherein said viralinfection is a HCV or HIV infection.
 35. A method according to claim 29wherein in step (ii) said test agent is administered to said animal. 36.A method according to claim 29 wherein (iii) or (iv) comprises one ormore of the following: (a) monitoring cell death; (b) monitoring viralreplication; (c) monitoring protein synthesis; and (d) monitoringpresence of viral protein on the surface of said cells.
 37. Ananti-viral vaccine or agent identified by a method according to claim29.
 38. Use of an antiviral vaccine or agent according to claim 69 inthe manufacture of a medicament for use in the therapeutic orprophylactic treatment of a viral infection.
 39. Use according to claim38 wherein said viral infection is a HCV or HIV infection.
 40. Themethod according to claim 28 wherein said viral infection is a HCV orHIV infection.
 41. The method according to claim 29 wherein said viralinfection is a HCV or HIV infection.